FUNCTION AND BIOLOGY OF EUKARYOTIC DNA TOPOISOMERASES

描述(改编自申请人的摘要)拓扑异构酶II是一种 正确的染色体结构所需的基本酶和 分离，并在DNA复制和重组中发挥重要作用。 除了其关键的细胞功能外，它还是一些 用于治疗人类疾病的最活跃和最广泛的处方药 癌症。这些药物通过一种机制来诱导它们的细胞毒作用 与其他药物有明显不同。而不是抑制 靶向抗癌药物拓扑异构酶II的催化活性 显著增加共价拓扑异构酶II裂解的DNA复合体水平 这些是正常的，但转瞬即逝的催化中间体。当所产生的 拓扑异构酶II相关的双链DNA断裂在高 浓度，它们会产生突变，染色体易位，以及 触发细胞死亡途径。 尽管拓扑异构酶II是癌症最重要的靶点之一 化疗，有令人信服的间接证据表明，这种酶还 有可能引发这种疾病。的确，继发性白血病 与特定的染色体易位相关的在一些 接受拓扑异构酶II靶向药物治疗的患者。因为拓扑异构酶II 必须在DNA中产生双链断裂才能发挥作用，这种酶 对基因组的完整性构成了内在的威胁 遗传物质。 尽管拓扑异构酶II对癌症问题至关重要， 该酶与DNA和抗癌药物的相互作用还不是很好 特色化的。因此，这项提案的最终目标是进一步划定 拓扑异构酶II执行其基本细胞功能的机制 反应和药物改变其催化功能的机制 酵素。这项研究的研究模型将是人类草履虫 小球藻病毒-1(PBCV-1)、酵母(酿酒酵母)和果蝇。 这项提案的具体目的是：1)进一步定义催化剂 拓扑异构酶的作用机制II；2)确定异常的基础 PBCV-1拓扑异构酶II的DNA切割活性；3)进一步描述 拓扑异构酶II靶向抗癌作用的机制基础 药物；4)探索拓扑异构酶II与基因组的关系 稳定性。拟议的实验是基于一些新的发现。 在上一个赠款周期期间作出的，包括发现PBCV-1 拓扑异构酶II，第一个真核病毒II型酶 特色化的。研究将极大地利用几种分析方法，这些方法是 在首席调查员的实验室开发，并将利用 生物化学、物理和遗传方法，以实现所述的目标 求婚。

DESCRIPTION (adapted from applicant's abstract) Topoisomerase II is an essential enzyme that is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical cellular functions, it is the primary target for some of the most active and widely prescribed drugs used for the treatment of human cancers. These agents elicit their cytotoxic effects by a mechanism that is markedly different than that of other drugs. Rather than inhibiting the catalytic activity of topoisomerase II, anticancer drugs targeted to the enzyme dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, catalytic intermediates. When the resulting topoisomerase II-associated double-stranded DNA breaks are present in high concentrations, they generate mutations, chromosomal translocations, and trigger cell death pathways. Although topoisomerase II is one of the most important targets for cancer chemotherapy, there is compelling circumstantial evidence that the enzyme also has the potential to trigger the disease. Indeed, secondary leukemias associated with specific chromosomal translocations are observed in some patients treated with topoisomerase II-targeted drugs. Because topoisomerase II must create double-stranded breaks in DNA in order to function, the enzyme poses an intrinsic threat to genomic integrity every time it acts on the genetic material. Despite the central importance of topoisomerase II to the cancer problem, interactions of the enzyme with DNA and anticancer drugs have not been well characterized. Thus, the ultimate goal of this proposal is to further delineate the mechanism by which topoisomerase II carries out its fundamental cellular reactions and the mechanism by which drugs alter the catalytic function of the enzyme. Research models for this study will be human, Paramecium bursaria Chlorella virus-1 (PBCV-1), yeast (Saccharomyces cerevisiae), and Drosophila. The specific aims of this proposal are to: 1) further define the catalytic mechanism of topoisomerase II; 2) determine the basis for the exceptionally robust DNA cleavage activity of PBCV-1 topoisomerase II; 3) further delineate the mechanistic basis for the actions of topoisomerase II-targeted anticancer drugs; and 4) explore relationships between topoismerase II and genomic stability. The proposed experiments are based on a number of novel findings made during the previous grant cycle, including the discovery of PBCV-1 topoisomerase II, the first eukaryotic viral type II enzyme to be characterized. Studies will take great advantage of several assays that were developed in the principal investigator's laboratory and will utilize biochemical, physical, and genetic approaches to address the stated aims of the proposal.